Brantley has family connections to the University of Florida. His father, John Brantley, III, also played quarterback there in the late 1970s and his uncle, Scot Brantley, played linebacker for the Gators and went on to the NFL.

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As a sophomore, Brantley threw for 1,201 yards, 17 touchdowns and one interception, while sharing time with senior Seth Varnadore. During his junior season, he threw for 2,835 yards, 41 touchdowns and five interceptions, leading Trinity Catholic to the Florida Class 2B state title. Brantley finished his high school career with an FHSAA record 99 touchdown passes, breaking the previous state mark of 98, held by Tim Tebow. Following his high school career, Brantley was invited to play in the 2007 U.S. Army All-American Bowl.

After a trip to Austin to see the University of Texas, Brantley committed to the Longhorns. In a later interview with FloridaFB.com, Brantley reiterated his commitment to Texas, stating that he felt it was "a real special thing to be headed out there [Texas]."

In December 2006, Rivals.com reported that Brantley would de-commit from Texas to follow in the footsteps of his uncle at the University of Florida. He later signed his letter of intent, and enrolled in Gainesville in 2007.

Brantley redshirted his first season with the Gators in 2007, before competing with Cam Newton for the backup job as a redshirt freshman in the 2008 season. Brantley first saw playing time in the season opener against Hawaii, and would go in as the backup after the starters were pulled in several conference games. He appeared in nine games, with pass attempts in seven of them. Brantley finished the 2008 season going 18 for 28 passing for 235 yards, three touchdowns, and one interception.

Brantley earned media attention during the 2009 season after starting quarterback Tim Tebow suffered a concussion in a September 26 game against Kentucky. It was speculated Brantley would make the first start of his Gator career in Baton Rouge at LSU, but Tebow was cleared to play on the day of the game.

In Florida's spring game of 2010, he completed 15 of 19 passes for 201 yards and two touchdowns.[4]

Brantley was the starting quarterback in the Gators' 2011 season. He injured his ankle in the October 1 game against Alabama and missed the following two games.